Ethanol production from lignocellulose

ABSTRACT

This invention presents a method of improving enzymatic degradation of lignocellulose, as in the production of ethanol from lignocellulosic material, through the use of ultrasonic treatment. The invention shows that ultrasonic treatment reduces cellulase requirements by ⅓ to ½. With the cost of enzymes being a major problem in the cost-effective production of ethanol from lignocellulosic material, this invention presents a significant improvement over presently available methods.

GOVERNMENT FUNDING

[0001] This invention was made, in part, with support by grants from theUnited States Department of Agriculture (95-37308-1843), the UnitedStates Department of Agriculture Cooperative Research Agreement(3620-41000-051-02S), the United States Department of Energy(DE-FG05-86ER13574) and the University of Florida. The government mayhave certain rights in the application.

BACKGROUND OF THE INVENTION

[0002] Ultrasound can be defined as sound waves above the range of humanperception (Price, 1992). Currently, many ultrasonic technologies suchas SONAR, medical-diagnostics, and surface cleaners are available. SONARand medical applications typically use low power and high frequency (≧1MHz). Surface cleaning applications, however, depend on ultrasoniccavitations created by lower frequency (20-50 Khz) and high powerultrasound.

[0003] Ultrasonic cavitations result from the rapid compression andexpansion of a liquid. In the expansion phase, the liquid is “tornapart”, resulting in the formation of voids or bubbles (Price, 1992;Leeman and Vaughan, 1992). These bubbles gradually increase in sizeuntil a critical size is reached, where critical size (usually 100-200μm in diameter) is dependent on the frequency of the oscillation and thepresence of any nucleating agents, e.g., dissolved gasses, cracks andcrevices on a solid surface, or suspended solids (Atchley and Crum,1988; Price, 1992). Once its critical size is reached, the bubbleimplodes, at times, generating temperatures approaching 5,500° C. withinthe bubble (Suslick, 1989, Price, 1992). When collapse of a cavityoccurs in a solution free of solid particles, heating is the onlyconsequence. However, if implosion occurs near a solid surface,implosion is asymmetric. As water rushes to fill the void left by theimploding bubble (e.g., at speeds near 400 m/s) shock pressures of 1-5Kpa can be generated (Suslick, 1988; Suslick, 1989; Price, 1992).

[0004] The physical effects of ultrasonic cavitations have been knownsince the early testing of the first British destroyer, the H.M.S.Daring, in 1894 (Suslick, 1990). The rapid revolution of a shippropeller creates the same, high frequency, compressions and expansionscreated by ultrasound (Suslick, 1989). Cavitations around the Daring'spropeller caused pitting of the metals used. This effect of cavitationson metal surfaces has been confirmed in studies on ultrasoniccavitations (Leeman and Vaughan, 1992; Boudjouk, 1988). High intensitystirring, the dispersal of suspended solids, increased diffusion throughcellulose gels, and emulsification of immiscible liquids are othereffects attributable to ultrasonic cavitations (Ensminger, 1973).

[0005] The high temperatures, pressures and velocities produced byultrasonic cavitation can also create unusual chemical environments(Suslick, 1989). Compounds in aqueous solution have been shown to formfree radicals when subjected to ultrasound. Water, when subjected toultrasound, creates H. and .OH intermediates, ultimately producing H₂and H₂O₂ (Suslick, 1988). Other chemical effects can be caused by highvelocity collisions driven by shock waves. The agglomeration of metallicparticles in ultrasonic fields has been shown (Suslick, 1989; Suslick,1990).

[0006] Ultrasonic surface cleaners have been available for use since theearly 1950's (Shoh, 1988). The mechanism of the cleaning action isreliant on the formation of cavitation bubbles. The contaminant coat canbe gradually eroded through cavitational action. Alternatively, theformation of cavitational bubbles between the coat and the surface,effectively peels the coat away from the surface. Other designs ofultrasonic cleaning systems have extremely high efficiency (>95%).

[0007] Most biological applications of ultrasonic technology have beendirected towards the disruption of cell membranes (Shoh, 1988; Ausubel,1996). One such device is Fisher Scientific's Model 550 SonicDismembrator. Recently, the effects of lower intensities of ultrasoundon bacteria have been investigated. It has been shown that nonlethaldoses of ultrasound may cause the induction of the SOS response and thetranscription of heat shock proteins in Escherichia coli (Volmer et al.,1996). Some of the physical damage to E. coli, by ultrasonic cavitation,has been illustrated recently (Allison et al., 1996), showing thedisruption of the plasma membrane and subsequent leakage ofintracellular components.

[0008] In the fermentation of milk by Lactobacillus bulgaricus, the rateof lactose hydrolysis was increased with the use of discontinuousultrasound (Wang et al., 1996). Presumably, the cause of the increasedrate of hydrolysis was the release of intracellular enzymes into themedia. After ultrasonic treatment was stopped, L. bulgaricus was able torecover and grow.

[0009] Recent interest in ultrasound has been shown by those involved inresearch in the paper industry investigating its uses as a de-inkingdevice in the recycling of various office paper (Scott and Gerber, 1995;Sell et al., 1995; Norman et al., 1994). It was reported that, becauseof ultrasonic treatment, the structure of the paper was changed suchthat its water holding capacity increased.

[0010] Besides the recycling of paper products, there is an interest inthe fermentation of waste paper and other lignocellulosic products intoethanol. The production of ethanol from such products reducesenvironmental waste problems and reduces reliance on petroleum-basedautomotive fuels. (Hohmann and Rendleman, 1993; Sheehan, 1993).Accessibility of the substrate to cellulase is a primary factorinfluencing the efficiency of enzymatic degradation of cellulose (Nazhadet al., 1995).

[0011] Cellulase from T. longibrachiatum is known to bind to cellulosetightly (Brooks and Ingram, 1995). The binding has also been shown to bedependent on the intensity of agitation (Kaya et al., 1994). Similareffects were seen with an intensive mass transfer reactor, whereextremely high rates of hydrolysis were achieved (Gusakov et al., 1996).

SUMMARY OF THE INVENTION

[0012] Improved methods for enzymatically converting lignocellulose, forexample, to ethanol, are desirable. This invention reports the use ofultrasonic treatment in a Simultaneous Saccharification and Fermentation(SSF) process to enhance the ability of cellulase to hydrolyze mixedoffice waste paper (MOWP), thereby reducing cellulase requirements by ⅓to ½. SSF is a process wherein ethanologenic organisms, such asgenetically engineered micro-organisms, such as Escherichia coli KO11(Ingram et al., 1991) and Klebsiella oxytoca P2 (Ingram et al., 1995),are combined with cellulase enzymes and lignocellulose to produceethanol. Enzyme cost is a major problem for all SSF processes.

[0013] In conducting the invention, enzyme stability is not affectedand, surprisingly, continuous ultrasonic treatment results in a decreasein hydrolysis relative to discontinuous treatment. One possibleexplanation is that the resultant mixing does not allow the cellulase torebind cellulose long enough for catalysis to occur. Therefore, time toallow catalysis between ultrasonic treatments is desired.

[0014] The SSF of waste office paper by K. oxytoca may also be “cycledependent.” Considering the inhibitory effect of ultrasound on thegrowth of K. oxytoca P2, a “recovery period” appears to be desired. Withvariation in the treatment schedule, such as increasing or decreasingtreatment time throughout the course of fermentation, furtheroptimization of the fermentation can be possible.

[0015] The use of ultrasound in the conversion of cellulose to ethanolrepresents a significant improvement in the SSF process. This isparticularly true where lignin residues were used to generate theelectricity required for the process. Ultrasound can be delivered inother manners as well, with liquid whistle systems, which are able toincrease the water holding capacity of recycled paper (Scott and Gerber,1995). Such a device in a piping loop can produce the desired disruptionof the fine structure of cellulose, with a lower energy input.

[0016] In one embodiment, the invention comprises a method for theenzymatic degradation of lignocellulose, such as in the production ofethanol from lignocellulosic material, comprising subjecting thematerial to ultrasound, as in a continuously-operating ultrasonicdevice, cellulase enzymes, optionally an ethanologenic yeast or anethanologenic bacterium and/or a fermentable sugar, and maintaining themixture thus formed under conditions suitable for the production ofethanol. In an alternative embodiment, the ultrasonic device is operateddiscontinuously.

[0017] In a preferred embodiment, the ethanologenic organisms areorganisms (particularly recombinant bacteria or yeast) which express oneor more enzymes or enzyme systems which, in turn, catalyze (individuallyor in concert) the conversion of a sugar (e.g., xylose and/or glucose)to ethanol. Preferred ethanologenic organisms include species ofZymomonas, Erwinia, Klebsiella, Xanthomonas and Escherichia. In a highlypreferred embodiment, the bacterium is K. oxytoca P2.

[0018] In another embodiment the ethanologenic yeast or ethanologenicbacterium contains enzymes that degrade lignocellulosic material,wherein the enzymes are released from the ethanologenic micro-organismby ultrasonic disruption.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows that ultrasonic treatment did not affect the activityof the added cellulase or β-glucosidase.

[0020]FIGS. 2A and 2B show the susceptibility of K. oxytoca P2 toultrasonic damage.

DETAILED DESCRIPTION OF THE INVENTION

[0021] As described above, the invention relates to an improved methodfor the enzymatic hydrolysis of lignocellulose comprising subjecting anaqueous mixture containing lignocellulose with ultrasound; andcontacting the mixture with a cellulase under conditions sufficient forhydrolysis. The aqueous mixture can be subjected to the ultrasoundtreatment continuously or discontinuously. Typically, the ultrasoundwill be conducted with commercially available equipment Examples ofsuitable ultrasonic probes include the RS-20 Ultrasonic TubularResonator and the RG-36/RS-36 Tube Resonator Systems (Telsonic USA,Bridgeport, N.J.). These ultrasonic probes may be combined withultrasonic generators to maintain desired operating parameters, such asoperating frequency and power. Examples of suitable ultrasonicgenerators include the RG-20 Ultrasonic-Generator and the MRG-36-150Module-Cleaning-Generator (Telsonic USA, Bridgeport, N.J.). Theultrasound treatment may be conducted at a wide-range of frequencies,all of which exhibit similar effects. For example, the frequency can bebetween above 2 and 200 kHz. The duration and conditions of theultrasonic step is selected to avoid overheating of the mixture to atemperature at which significant amounts of the enzyme(s) will bedenatured. Generally, the duration of the ultrasound treatment lastsbetween 10 minutes and 30 minutes. Without being limited in anyway bytheory, the ultrasound treatment is typically sufficient to disrupt thecrystalline structure of the lignocellulosic material.

[0022] The term “continuous” treatment is defined herein to include asingle treatment with ultrasound for the duration of the enzymatichydrolysis, i.e. there are no intermediary periods between or duringenzymatic hydrolysis in which there is no ultrasound. The term“discontinuous” treatment is defined herein to include multipletreatments with ultrasound between or during enzymatic hydrolysis. Inyet another embodiment, the ultrasonic treatment can be a singleexposure to ultrasound prior to enzymatic hydrolysis.

[0023] The lignocellulose material can be obtained from lignocellulosicwaste products, such as plant residues and waste paper. Examples ofsuitable plant residues include stems, leaves, hulls, husks, cobs andthe like, as well as wood, wood chips, wood pulp, and sawdust. Examplesof paper waste include discard photocopy paper, computer printer paper,notebook paper, notepad paper, typewriter paper, and the like, as wellas newspapers, magazines, cardboard, and paper-based packagingmaterials.

[0024] The aqueous mixture containing lignocellulose subjected to theultrasonic treatment can further comprise a cellulase enzyme for theenzymatic hydrolysis. In yet another embodiment, the cellulase enzyme isadded subsequent to the ultrasound treatment. The cellulase can beprovided as a purified enzyme or can be provided by acellulase-producing microorganism in said aqueous mixture. Cellulases,as that term is used herein, includes any enzyme that effects thehydrolysis or otherwise solubilizes cellulase (including insolublecellulose and soluble products of cellulose). Cellulase enzymes,including purified enzyme preparations, organisms expressing the same,are known in the art. Suitable sources of cellulase include suchcommercial cellulase products as Spezyme™ CP, Cytolase™ M104, andMultifect™ CL (Genencor, South San Francisco, Calif.), and suchorganisms expressing cellulase as the recombinant bacterium of U.S. Pat.No. 5,424,202, which is incorporated herein by reference.

[0025] The conditions for cellulase hydrolysis are typically selected inconsideration of the conditions suitable for the specific cellulasesource, e.g, bacterial or fungal. For example, cellulase from fungalsources typically works best at temperatures between about 30° C. and48° C. and a pH between about 4.0 and 6.0. In general, typicalconditions include a temperature between about 30° C. and 60° C. and apH between about 4.0 and 8.0.

[0026] The aqueous mixture can further advantageously comprise anethanologenic microorganism which has the ability to convert a sugar oroligosaccharide to ethanol. Ethanologenic microorganisms are known inthe art and include ethanologenic bacteria and yeast. The microorganismsare ethanologenic by virtue of their ability to express one or moreenzymes which, individually or together, convert a sugar to ethanol. Itis well known, for example, that Saccharomyces (such as S. cerevisiae)are employed in the conversion of glucose to ethanol. Othermicroorganisms that convert sugars to ethanol include species ofSchizosaccharomyces (such as S. pombe), Zymomonas (including Z.mobilis), Pichia (P. stipitis), Candida (C. shehatae) and Pachysolen (P.tannophilus).

[0027] Preferred examples of ethanologenic microorganisms includeethanologenic microorganisms expressing alcohol dehydrogenase andpyruvate decarboxylase, such as can be obtained with or from Zymomonasmobilis (see U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; and5,482,846, all of which are incorporated herein by reference).

[0028] In another embodiment, the ethanologenic microorganism canexpress xylose reductase and xylitol dehydrogenase, which convert xyloseto xylulose. Xylose isomerase converts xylose to xylulose, as well. Theethanologenic microorganism can further express xylulokinase, whichcatalyzes the conversion of xylulose to xylulose-5-phosphate. Additionalenzymes to complete the pathway can include transaldolase andtransketolase. These enzymes can be obtained or derived from Escherichiacoli, Klebsiella oxytoca and Erwinia species. For example, see U.S. Pat.No. 5,514,583.

[0029] It is particularly preferred to employ a microorganism which iscapable of fermenting both pentoses and hexoses to ethanol, such as areobtained from preparing a recombinant organism which inherentlypossesses one set of enzymes and which is genetically engineered tocontain a complementing set of enzymes. Examples of such microorganismsinclude those described in U.S. Pat. Nos. 5,000,000; 5,028,539;5,424,202; 5,482,846; 5,514,583; and Ho et al., WO 95/13362, all ofwhich are incorporated herein by reference. Particularly preferredmicroorganisms include Klebsiella oxytoca P2 and Escherichia coli KO11.

[0030] The conditions for converting sugars to ethanol are typicallythose described in the above referenced U.S. Patents. Generally, thetemperature is between about 30° C. and 40° C. and the pH is betweenabout 5.0 and 7.0.

[0031] It is generally advantageous to add nutrients and/or cofactorsfor the microorganisms and/or enzymes to optimize the enzymaticconversions. For example, xylose reductase employs NADPH and xylitoldehydrogenase employs NAD as cofactors for their respective enzymaticactions. In contrast, bacterial xylose isomerase requires no co-factorfor direct conversion of xylose to xylulose. It is also desirable toadd, or subject the microorganism separately to, assimilable carbon,nitrogen and sulfur sources to promote growth. Many mediums in which togrow microorganisms are well known in the art, particularly Luria broth(LB) (Luria and Delbruk, 1943).

[0032] Where the ultrasound treatment is conducted in the presence of amicroorganism, the ultrasound can be conducted at a frequency andduration such that a portion of all the microorganisms present are lysedor otherwise subjected to membrane disruption. Such a method can resultin a controlled release of the enzymes from the microorganisms into thesurrounding medium, thereby allowing the optimization of enzymes eitheralone or in conjunction with commercial enzymes and reduce the overallcost of commercial enzymes.

[0033] Examples of microorganisms containing desirable enzymes includethose described in U.S. Pat. No. 5,424,202 to Ingram, et al. Othermicroorganisms are disclosed in U.S. Pat. Nos. 5,028,539 to Ingram etal., 5,000,000 to Ingram et al., 5,487,989 to Fowler et al., 5,482,846to Ingram et al., 5,554,520 to Fowler et al., 5,514,583 to Picataggio,et al., copending applications having U.S. Ser. Nos. 08/363,868 filed onDec. 27, 1994, 08/475,925 filed on Jun. 7, 1995 and 08/218,914 filed onMar. 28, 1994 and standard texts such as, Ausubel et al., CurrentProtocols in Molecular Biology, Wiley-Interscience, New York (1988)(hereinafter “Ausubel et al.”,), Sambrook et al., Molecular Cloning: ALaboratory Manual, Second and Third Edition, Cold Spring HarborLaboratory Press (1989 and 1992) (hereinafter “Sambrook et al.”) andBergey's Manual of Systematic Bacteriology, William & Wilkins Co.,Baltimore (1984) (hereinafter “Bergey's Manual”) the teachings of all ofwhich are hereby incorporated by reference in their entirety. Yet otherembodiments include those described in U.S. Ser. No. ______, filedconcurrently herewith by Ingram et al. (Attorney Docket No. UF97-01) andU.S. Ser. No. ______ by Ingram et al. (Attorney Docket No. UF97-02),which are incorporated herein by reference.

[0034] An example of a suitable device to deliver the ultrasound isFisher Scientific's Model 550 Sonic Dismembrator, Telsonic UltrasonicTubular Resonator RS-20, Telsonic Ultrasonic-Generator RG-20, orTelsonic Tube Resonator System Series RG-36/RS-36. In one embodiment, anultrasonic immersion horn can be used directly in the aqueous medium.Alternatively, the ultrasound can be emitted into a liquid filled vat incontact with a vat containing the aqueous medium (such as a first vatplaced within a second vat, either of which can contain the aqueousmedium). It may also be desirable, in a continuous system to flow theaqueous medium through a container, or vat, with the ultrasonic devicewhich, continuously or discontinuously, emits ultrasound. In yet anotherembodiment, it can be desirable to control the temperature of theaqueous medium by surrounding the container, or vat, with cooling water,or other suitable heat exchange arrangement. It is within the ability ofone of ordinary skill in the art to determine how to optimize therelease of enzymes from microorganisms, said enzymes to be used alone orin conjunction with commercial enzymes, to achieve optimum ethanolproduction.

[0035] Methods and Materials

[0036] The methods and materials described below were used in carryingout the work described in the examples which follow. For convenience andease of understanding, the methods and materials section is divided intosub-headings as follows.

[0037] Organism and Media

[0038] All fermentations of Mixed Waste Office Paper (MWOP) used K.oxytoca P2 as the biocatalyst. Luria broth (LB) (Luria and Delbruk,1943) was used as the source of nutrients for all liquid and solidmedia. Solid media also contained 15 g/L agar and 20 g/L glucose. Forthe propagation of inoculum, liquid media containing 50 g/L glucose wasused. Chloramphenicol (40 mg/L) was used as required for selection.Cultures were maintained on agar plates containing either 40 mg/L Cm or600 mg/L Cm. The commercial cellulase Spezyme™ CP (Genencor, South SanFrancisco, Calif.), a mixture of cellulase enzymes from Trichodermalongibrachiatum (formerly T. reesei), was used. Novozyme 188,β-glucosidase from Aspergillus niger (Novo-Nordsk, Franklintin N.C.) wasalso used in saccharification experiments.

[0039] Enzyme Activity and Sugar Analysis

[0040] Endoglucanase activity was determined as previously described(Wood and Bhat, 1988). Cellulase mixtures were diluted in 50 mM citratebuffer, pH 5.2, containing 2% CMC and incubated at 35° C. Release ofreducing sugars was determined by the DNS method as described (Chaplin,1987). Cellobiase activities were determined by measuring the rate ofp-nitrophenol (p-NP) release (Abs._(410 nm)) fromp-nitrophenyl-β-D-glucoside (p-NPG) at pH 5.2, 35° C. (Wood and Bhat,1988). Enzyme solutions were diluted in 50 mM citrate buffer, pH 5.2, asrequired. One ml of diluted enzyme was added to 1 ml 2 mM p-NPG andincubated at 35° C. Reactions were terminated with the addition of 1 MNa₂CO₃.

[0041] Enhancement of Sugar Release from MWOP

[0042] 125 g dry wt. shredded MWOP was added with 25 ml 18 N H₂SO₄ and 2L H₂O in a 3-liter stainless steel beaker. The slurry was allowed toreact fully with the carbonate present in the paper (monitored by gasevolution). The pH was then adjusted to approximately 2.5 and autoclaved(121° C.) for 20 minutes. After overnight cooling, 125 ml 1 M sodiumcitrate was added and the volume was brought to 2.5 L with H₂O. The pHwas adjusted to 5.2 and was placed in a constant temperature bath, 35°C. Mixing was done with a 750-mm Rushton-type radial flow impeller and aCiambanco model BDC-1850 laboratory mixer. Five FPU Spezyme™ CP per gramof paper (625 FPU/2.5 L) and 50 U Novozyme 188 per liter (250 U/2.5L)were also added. Units used were as reported by the manufacturer.Thymol, 0.5 g/L, and chloramphenicol, 40 mg/L was added to preventmicrobial growth. Ultrasound was produced by a Telsonic 36 KHz TubeResonator (>95% efficiency), model RS-36-30-1 with an accompanying modelMRG-36-150 (150 W effective output) ultrasonic generator (Telsonic USA,Bridgeport, N.J.). The frequency was tuned automatically. Treatmentcycles were controlled by an SPER Scientific 810030 timer (FisherScientific Co., St. Louis, Mo.). Mixing speeds were constantly adjustedto the lowest setting that would allow mixing (600-75 rpm).

[0043] Enzyme Stability

[0044] The enzyme preparations were diluted in 50 mM citrate buffer toconcentrations equivalent to those used in the study of sugar releasefrom MWOP, 250 FPU Spezyme™ CP/L and 50 U/L Novozyme 188. Solutions alsocontained 0.5 g/L thymol and 40 mg/L Cm to prevent microbial growth. Theenzyme mixture was stirred (120 RPM) for 15 minutes to ensure completedispersal of the enzyme. Stirring was continued for 48 hours with orwithout continuous exposure to ultrasound. Samples were taken at 0, 12,24, 36, and 48 hours. Enzyme activities were assayed as described above.

[0045] Cell Viability

[0046] To 1.75 L of LB containing 50 g/L glucose and 40 mg/L Cm, K.oxytoca P2 was used to inoculate to an initial cell density, measured asO.D.₅₅₀ nm, of 0.5. Growth was allowed to proceed for 12 hours with orwithout ultrasonic treatment. Samples were taken and dilutions were madeto follow cell growth at 0, ¼, ½, 1, 2, 4, 8, and 12 hours. Opticaldensity (O.D.550 nm) and pH were measured on each sample. Dilutions werespread on agar plates (20 g/l glucose) and incubated overnight (30+C.).Colony forming units (CFUs) were mounted as a measurement of cellviability.

[0047] Ultrastructural Effects

[0048] The change in the structure of the cellulose matrix of MWOP wasinvestigated using a Hitachi S4000 scanning electron microscope. Sampleswere prepared by subjecting 2.5 L mixtures of 50 g/L MWOP in 50 mMcitrate buffer, pH 5.2 and 35° C., to one hour of continuous ultrasound.Other samples were treated with cellulase for 4 hours. Control sampleswere taken before any treatment. All samples were dried and sputtercoated with gold before being examined (Doran et al., 1994).

[0049] Cell Propagation

[0050]K. oxytoca P2 was transferred from a stock culture (−20° C.) toagar plates with 20 g/L glucose and Cm (40 mg/l and 600 mg/l). Anisolated colony was then transferred daily from the plate with 600 mg/LCm to fresh plates containing both concentrations of Cm. Isolatedcolonies from plates with 40 mg/L Cm were used to inoculate flasks withLB and 50 g/L glucose. Inoculated flasks were incubated overnight at 30°C. after which they were harvested by centrifugation for further use.

[0051] SSF with Ultrasonic Treatment

[0052] Fermentations of MWOP were conducted in 14 L glass fermentationvessels (10 L working volume) using Multiferm™ fermentors models 100 and200 (New Brunswick Sci. Co., NJ). Stainless steel head plates weremodified by removing components that extended into the broth. Headplates were sanitized with 10 g/L formaldehyde by coating all surfaceswith the formaldehyde while loosely enclosed in a large plasticautoclave bag. One kg, dry weight, shredded MWOP was placed infermentation vessels with 8 L H₂O and 110 ml 18 N H₂SO₄. The mixture wasautoclaved for one hour. After cooling, the slurry was furtherhomogenized by vigorous mixing with a hand drill and a paint mixingattachment. After autoclaving for an additional one hour and subsequentcooling, 5 FPU Spezyme™ CP/g MWOP, 1 L 10× LB (pH 5.0) and H₂O was addedto a final volume of 10 L. This solution was partially mixed by hand,using a sterilized industrial baking whisk, to disperse the enzymes andnutrients. Cells were added to an initial O.D.₅₅₀ nm of 0.5. Ultrasonictreatments were as described above. Because of its nonhomogeneousnature, no samples were taken for an initial ethanol determination.Samples were taken at 24, 48, 72, and 96 hours.

EXAMPLE 1 Analysis of Enhanced Rates of Sugar Release

[0053] Using the methods and materials outlined above for “Enhancementof Sugar Release from MWOP,” it was found that with the use ofultrasonic energy the rate of enzymatic hydrolysis was increased up to40%. When sugar release with ultrasonic treatment 15 minutes every fourhours is compared with treatment every two hours,a strong correlationbetween the amount of ultrasonic energy and sugar release is found. Theincreased rate of sugar release is due to a stimulation of enzymaticactivity, not a physical or chemical hydrolysis by reactive byproductsfrom the sonolysis of water, as illustrated by the experiments withoutenzymes added. Interestingly, with continuous ultrasonic treatment, therate of the hydrolysis goes down. Results are set forth in table formatin Table 1. TABLE 1 Effects of ultrasonic cavitation on enzymatichydrolysis of mixed waste office paper. Energy Glucose equivalents^(b,c)Ultrasonic Number of input (mM) treatment^(a) Experiments (W) @ 24 h 36h 48 h No ultrasound 3 0 88.1 ± 6.1 98.3 ± 6.1 106.9 ± 7.8  15 min. Per4 h 3 9.37 96.5 ± 6.4 113.6 ± 8.0  128.3 ± 8.4  15 min. Per 2 h 3 18.75115.5 ± 14.3 133.2 ± 10.6 149.0 ± 11.0 Continuous 3 150 98.1 ± 2.5 112.5± 5.5  126.6 ± 2.1  Continuous (no 2 150 0.67 0.67 0.58 enzyme)

EXAMPLE 2 Enzyme Stability

[0054] Using the methods and materials outlined above for “EnzymeStability,” it was found that ultrasonic treatment did not affect thestability of the added cellulase or β-glucosidase, as depicted inFIG. 1. Both activities remained quite stable even with continuousexposure to ultrasound. The apparent increase in β-glucosidase may bedue to the dispersal of protein aggregates in the highly concentrated,commercial, enzyme preparation.

EXAMPLE 3 Cell Viability

[0055] Using the methods and materials outlined above for “CellViability,” it was found that ultrasonic treatment appeared to benonlethal, but was inhibitory to growth, as shown in FIGS. 2A and 2B.This observation may be due in part to an induction of an SOS responseby the cells. This was further supported by the observations of pH,which slightly increased (pH 6.9 from an initial pH 6.7). Additionally,it was observed that the relative turbidity of the broth had littlechange throughout the exposure to ultrasound. Meanwhile, withoutultrasonic treatment, a classical growth curve was observed.

EXAMPLE 4 Effects on SSF

[0056] Using the methods and materials outlined above for “SSF withUltrasonic Treatment,” the combination of K. oxytoca P2 with ultrasonictreatment resulted in as much as a 15% increase in ethanol yields.Ethanol production from waste office paper treated with ultrasound andK. oxytoca P2 is summarized in Table 2. As might be expected from theinhibition of cell growth, increased ultrasonic treatment results inreduced ethanol production. Treatment every two hours may not besignificantly different from treatment every four hours, however, astatistically significant difference between ultrasonic treatment everyfour hours and no treatment was found. TABLE 2 Effects of ultrasonictreatment on ethanol production in SSF of MWOP using K. oxytoca P2 asthe biocatalyst [Enzyme]^(a) [Ethanol] Yield^(b,c) Ultrasonic (FPU/g(g/L) (GE/g treatment Replicates MWOP) 24 h 48 h 72 h 96 h Cellulose)None 2 10 15.7 27.3 33.5 35.3 0.39 None 4 5 9.5 ± 2.3 19.0 ± 2.7 25.7 ±2.5 29.4 ± 2.9 0.33 15 min. Per 4 h 5 5 14.3 ± 2.0 26.1 ± 1.3 31.3 ± 1.334.0 ± 1.9 0.38 (9.37 W) 15 min per 2 h 2 5 13.4 23.4 28.8 31.4 0.35(18.75 W) Continuous 2 5 10.2 11.2 11.3 11.3 0.13 (150 W)

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EQUIVALENTS

[0083] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is:
 1. A method for enzymatically degradinglignocellulose comprising the steps of: (a) subjecting an aqueousmixture containing lignocellulose with ultrasound; and (b) contactingthe mixture with a cellulase under conditions sufficient for hydrolysis.2. The method according to claim 1 wherein said aqueous mixture of step(a) further comprises said cellulase.
 3. The method according to claim 2wherein said cellulase is provided by a cellulase-producingmicroorganism in said aqueous mixture.
 4. The method according to claim2 wherein said step (a) is continuous.
 5. The method according to claim2 wherein said step (a) is discontinuous.
 6. The method according toclaim 1 wherein said ultrasound is conducted at a frequency of betweenabout 2 and 200 kHz.
 7. A method for enzymatically degradinglignocellulose comprising the steps of: (a) subjecting an aqueousmixture containing lignocellulose with ultrasound; and (b) contactingthe mixture with a cellulase and ethanologenic microorganism underconditions sufficient for hydrolysis.
 8. The method according to claim 7wherein said aqueous mixture of step (a) further comprises saidcellulase and ethanologenic microorganism.
 9. The method according toclaim 8 wherein said cellulase is provided by a cellulase-producingmicroorganism in said aqueous mixture.
 10. The method according to claim8 wherein said step (a) is continuous.
 11. The method according to claim8 wherein said step (a) is discontinuous.
 12. The method according toclaim 8 wherein said ultrasound is conducted at a frequency of betweenabout 2 and 200 kHz.
 13. The method according to claim 8 wherein saidethanologenic microorganism is an ethanologenic bacteria or yeast. 14.The method according to claim 13 wherein said ethanologenicmicroorganism is a bacteria or yeast which expresses one or more enzymeswhich, individually or together, convert a sugar to ethanol.
 15. Themethod according to claim 13 wherein said ethanologenic microorganismexpresses enzymes which, individually or together, convert pentose andhexose to ethanol.
 16. The method according to claim 13 wherein saidethanologenic microorganism expresses alcohol dehydrogenase and pyruvatedecarboxylase.
 17. The method according to claim 16 wherein said alcoholdehydrogenase and pyruvate decarboxylase are from Zymomonas mobilis. 18.The method according to claim 13 wherein said ethanologenicmicroorganism expresses xylose isomerase, xylulokinase, transaldolase,and transketolase.
 19. The method according to claim 18 wherein saidxylose isomerase, xylulokinase, transaldolase, and transketolase arefrom Escherichia coli.
 20. The method according to claim 18 wherein saidxylose isomerase, xylulokinase, transaldolase, and transketolase arefrom Klebsiella oxytoca.
 21. The method according to claim 18 whereinsaid xylose isomerase, xylulokinase, transaldolase, and transketolaseare from Erwinia species.
 22. The method according to claim 13 whereinsaid ethanologenic microorganism expresses alcohol dehydrogenase,pyruvate decarboxylase, xylose isomerase, xylulokinase, transaldolase,and transketolase.
 23. The method according to claim 22 wherein saidethanologenic microorganism is a recombinant microorganism expressingZymomonas mobilis alcohol dehydrogenase and pyruvate decarboxylasewherein said microorganism is selected from the group consisting ofEscherichia coli, Klebsiella oxytoca, and Erwinia species.
 24. Themethod according to claim 23 wherein said ethanologenic microorganism isKlebsiella oxytoca P2.
 25. The method according to claim 23, whereinsaid ethanologenic microorganism is Escherichia coli KO11.